R. D. Tee

The role of cereal and fungal amylases in cereal flour hypersensitivity

Abstract. To investigate the role of cereal α. and β‐amylase in bakers’ asthma, we have compared the IgE response of 30 wheat‐flour‐allergic individuals to barley α and β‐amylases with that of fungal α‐amylase using radioallergosorbenl test (RAST), RAST inhibition assays and Western blotting. RAST analysis showed 29 of the 30 subjects with inhalant induced cereal allergy had positive IgE to cereal amylases, but only 16 were positive to fungal α‐amylase. Regression analysis showed an association between specific IgE to wheat‐flour and to barley α‐amylase (r = 0.70) and barley β‐amylase (r= 0.92) but a poor association with fungal α‐amylase (r = 0–34). RAST inhibition showed minimal crossreactivity between barley α or β‐amylase and barley and fungal α‐amylase. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blotting showed that non‐reduced barley α‐amylase had a molecular weight of 54 kDa and barley β‐amylase a molecular weight of 64 kDa. Reduced fungal α‐amylase had a molecular weight of 54 k Da. Cereal α and β‐amylase appear to be important allergens in patients with allergy to flour.

Allergy to storage mites

Summary. There is now much evidence of sensitization to storage mites in urban populations as well as in the well‐documented rural populations. Sensitization is therefore not restricted only to those with occupational exposure.

Identification of crossreacting wheat, rye, barley and soya flour allergens using sera from individuals with wheat-induced asthma

We have conducted radio allergosorbent test (RAST), competitive RAST inhibition, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blotting using sera from patients with wheat‐induced asthma to investigate the immunological relationship between wheat, rye, barley and soya, and to identify common proteins between these flours. RAST showed strong associations between the levels of specific IgE to wheat flour and those of rye and barley flour. Competitive RAST inhibition showed that wheat, rye, barley and soya flours contained crossreacting proteins, in decreasing concentrations. Wheat, rye and barley flours had similar protein profiles on gel electrophoresis. Soya flour contained a number of high molecular weight proteins not present in the other cereals. Western blotting using sera from 21 wheat flour hypersensitive individuals identified a large number of allergens in the different flours. Proteins of 69, 33, 26, 21 and 12 kDa were identified as major wheat flour allergens. Rye flour proteins of 21 and 12 kDa, and barley flour proteins of 69, 52 and 10 kDa were the major allergens identified by serum from wheat hypersensitive individuals. The major common protein of soya and wheat flour had a molecular weight of 21 kDa. The majority of crossreacting allergens identified between the different flours have molecular weights similar to those of known flour enzymes or enzyme inhibitors.

Measurement of airborne rat urinary allergen in an epidemiological study

The suitability of radioallergosorbent test (RAST) inhibition to quantify occupational exposure to rat urinary aeroallergen (RUA) has been assessed. When using a constant pool of rat allergic sera, the reproducibility of the assay over 1 year was comparable to that reported for other immunoassays; at 50% RAST inhibition the inter‐assay coefficient of variation (CV) was 7.0% and the intra‐assay CV was 3.0%. The assay was highly specific for rat urine; mouse urine was 1100‐fold less potent at inhibiting the rat urine RAST system. Significant inter‐assay variation in the ‘high’ control was not due to batch variation and was relatively small when compared with the variation in RUA concentrations in the occupational environment. Measurement of workplace RUA exposure demonstrated that those directly involved in the care of rats experienced the highest RUA exposure of the nine occupational groups studied (animal technicians GM = 23.10/μg/m3) followed in decreasing order by those working with soiled litter (e.g. cage cleaners GM = 4.20 μg/m3), dead animals (e.g. post mortem GM = 1.60 μg/m3, scientists GM = 0.67 μg/m3) and rat tissue (e.g. slide production GM = 0.04μg/m3). In view of the complexity of rat allergens, RAST inhibition is an appropriate method for the quantification of occupational exposure to rats.

Specific immunological and bronchopulmonary responses following intradermal sensitization to free trimellitic anhydride in guinea pigs

We have developed a guinea pig model of trimellitic anhydride‐induced airway hypersensitivity responses. In one group of guinea pigs, injected intradermally with 0.1 ml 30% trimellitic anhydride (TMA), we examined the specificity of the bronchopulmonary response to TMA comparing the effect of intravenous TMA conjugated to guinea pig serum albumin (GPSA) with a control hapten (procion dye) protein conjugate (PD‐GPSA). A significant increase in pulmonary inflation pressure (PIP) was provoked in sensitized animals following intravenous injection with TMA‐GPSA (20%; 0–400, median; range) as compared to intravenous injection of PD‐GPSA. In the second group we compared three different methods of sensitization: single injection of 0.1 ml of 0.3% TMA; four injections of 0.1 ml of 0.1% TMA; and a single high dose injection of 30% TMA. Following intravenous TMA‐GPSA guinea pigs sensitized with a single injection 0.3% TMA had an increase in PIP of 395%; 220–600, while those given four repeat injections of 0.1% TMA had an increase in PIP of 343%; 315–490. These results were significantly higher than the increase in PIP (160%; 0–220) which occurred in guinea pigs sensitized with a single dose of 30% TMA. Four of 11 guinea pigs given low dose injections of TMA had bronchopulmonary responses to inhaled TMA‐GPSA. All sensitized guinea pigs had specific IgG1 antibodies demonstrated by enzyme linked immunosorbent assay (ELISA) and confirmed by ELISA inhibition. Four guinea pigs sensitized by low dose injections of TMA had IgE antibodies demonstrated by passive cutaneous anaphylaxis.

PEAK EXPOSURE CONCENTRATIONS OF DUST AND FLOUR AEROALLERGEN IN FLOUR MILLS AND BAKERIES

As part of an epidemiological study amongst workers exposed to flour we measured peak exposure levels to total dust and flour aeroallergen with personal samplers in bakeries, flour mills and packing stations. Short-term tasks which were expected to give rise to high concentrations of exposure (peaks) were identified. The frequency and duration of these tasks were estimated and their levels of exposure to dust and flour aeroallergen measured. In total 209 samples were taken. The highest exposure concentrations both for dust (geometric mean > 30 mg m-3) and for flour aeroallergen (geometric mean > 500 micrograms m-3) were measured during certain operations. Exposure concentrations for the tasks were often much higher than the levels we had measured over a shift in a previous study. This might be important for sensitization and for the development of asthma. Peak exposure concentrations could be used to explore the exposure-response relationship more comprehensively. In general average flour aeroallergen concentrations increased linearly with average dust concentrations, although there were some exceptions.

Measurement of airborne proteins involved in Bakers’ asthma

Abstract. We have developed a competitive inhibition immunoassay to measure airborne flour, using purified polyclonal rabbit IgG antibodies to cereal flour antigens. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) showed different flours contained proteins of molecular weight similar to those present in air sample eluates. Western blotting demonstrated that the purified rabbit IgG antibodies bound to proteins whose molecular weights are similar to human allergens. Air sample eluates obtained from a large bakery had slopes of inhibition parallel to cereal flour when compared in the immune‐inhibition assay. The assay identified airborne proteins from several flours with a detection limit of I μg ml‐1. Using this immunoassay we have shown that a wide range of airborne flour concentrations occur in flour mills and a smaller range bakeries. This assay which allows measurement of the concentration of Hour aeroallergen in the workplace will be valuable both in epidemiological studies and in examining the effectiveness of interventions to reduce the concentration of flour in the air.

Analysis of the allergenic composition of rat dust

Background Allergy to rats is an important occupational health problem. The allergens of rat urine have been well defined but those in rat room dust, a potentially important source of inhalant exposure, have not.

Variation in rat urinary aeroallergen levels explained by differences in site, task and exposure group

We describe how much of the variation in rat urinary aeroallergen (RUA) levels may be explained by differences in site and exposure group in research establishments and we identify task categories associated with high RUA levels. In this population 73% of the variability in RUA levels could be explained by exposure group, site and their interaction term. Exposure group alone explained the majority of the variation (69%). The task category associated with the highest RUA level was cleaning out, followed by indirect contact with rats and handling rats. The task categories associated with the lowest levels were slide production, post mortem and experiments. These results may help choose appropriate exposure measures for epidemiological studies in research establishments in the future and may help reduce the incidence of laboratory animal allergy.

Comparison of allergic responses to dust mites in U.K. bakery workers and Swedish farmers

The IgE RAST response to Dermatophagoides pteronyssinus and four storage mites (Lepidoglyphus destructor, Tyrophagus putrescentiae, Glycyphaghus domesticus, and Acarus siro) was examined in 251 U.K. bakery workers and compared with that previously found in 440 Swedish farmers. Storage mites are found commonly in stored hay and grain so both these groups potentially encounter them in their work. In neither group of workers was a positive RAST (≥0.35 PRU) to D. pteronyssinus correlated with a positive RAST to a single storage mite. As in the Swedish farmers, significant though not strong correlations were found in the U.K. bakers between positive RAST responses to G. domesticus and L. destructor and to T. putrescentiae and L. destructor (P < 0.05).